http://immunol.nature.com • august 2002 • volume 3 no 8 • nature immunology Stephan D. Gadola 1, * ,† , Nathan R. Zaccai 2, *, Karl Harlos 2 , Dawn Shepherd 1 , Julio C. Castro- Palomino 3 , Gerd Ritter 4 , Richard R. Schmidt 3 , E.Yvonne Jones 2 and Vincenzo Cerundolo 1 Published online: 15 July 2002, doi:10.1038/ni821 The human genome encodes five nonpolymorphic major histocompatibility complex class I–like glycoproteins, CD1a to CD1e, that present lipid antigens for specific recognition by T lymphocytes. Using single alkyl chain detergents, we developed a protocol to generate recombinant human CD1b- lipid complexes. We present here the crystal structures of CD1b in complex with either phosphatidylinositol or ganglioside GM2 at 2.3 Å and 2.8 Å resolutions, respectively. The antigen- binding groove houses four interlinked hydrophobic channels that are occupied by the alkyl chains of the glycolipid plus two detergent molecules. A distinct exit beneath the α2 helix further contributes to the plasticity of the binding groove. These structures reveal the mechanism by which two alkyl chain lipids bind to CD1b, and how CD1b can adapt to ligands of different alkyl chain length.They also suggest how very long alkyl chains, such as those of mycolic acid, could be fully contained within the binding groove. These results extend the spectrum of potential CD1b ligands by revealing that, in addition to two alkyl chain lipids, mono-alkyl and triple-alkyl chain lipids can be accommodated in the binding groove. 1 Cancer Research UK Tumour Immunology Group,The Weatherall Institute of Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DS, UK. 2 Cancer Research UK Receptor Structure Research Group,The Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Headington, Oxford OX3 7BN, UK. 3 Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany. 4 Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan Kettering Cancer Center, New York, NY, USA. *These authors contributed equally to this work. † Present address: Department of Rheumatology and Clinical Immunology, University of Berne, Inselspital, PKT2 D584, Berne CH- 3010, Switzerland. Correspondence should be addressed to S. D. G. (stephan.gadola@insel.ch), E.Y. J. (yvonne@strubi.ox.ac.uk) or V. C. (vincenzo.cerundolo@imm.ox.ac.uk). Structure of human CD1b with bound ligands at 2.3 Å, a maze for alkyl chains Human CD1 molecules segregate into two groups according to their sequence similarity 1 . Group 1 contains CD1a, CD1b, CD1c and CD1e molecules, whereas CD1d belongs to group 2. CD1a, CD1b and CD1c present mycobacterial phospho- and glycolipids as well as self-glycol- ipids, such as gangliosides, to specific T cells 2–6 . Based on in vitro bind- ing studies and T cell recognition assays, a common motif, consisting of a single proximal branched acyl chain or two acyl chains 2,6,7 , has been proposed for CD1a, CD1b and CD1d ligands, whereas CD1c presents isoprenoid glycolipids containing a single alkyl chain to T cells 8 . Several studies have clarified the intracellular trafficking pathways of the different CD1 iso- forms. The amino acid sequences of CD1b, CD1c and CD1d, but not of CD1a, contain a cytoplasmic tail motif for the recy- cling of cell surface–expressed CD1 molecules to intracellular compartments 9 . Although CD1b and CD1d localize mainly to late endosomes and lysosomes 10–12 , CD1c primarily accesses early endosomes 9 . Differential intracellular sorting of short and long alkyl chain CD1b ligands into nonendosomal and ARTICLES 721 Figure 1. Structure of the human CD1b-ligand complex. (a) CD1b structure (α1-α3 domains in greenish-blue, β2M in green) with bound PI (alkyl chains in yellow and red, phosphate in lilac, inos- itol in green) and detergent molecules (pink and violet) shown as Van Der Waals spheres.The internal hydrophobic cavity of α1α2 is drawn as a transparent surface, and ligands within the binding chan- nels are indicated as A′ (red), C′ (yellow), F′ (pink) and T′ (violet). (b) Chemical structures of lipid and detergent ligands used in the refold- ing of CD1b complexes. a b © 2002 Nature Publishing Group http://immunol.nature.com